Exploring the Sequential Cellular Events of Phagocytosis Triggered by Godanti Bhasma in Mammalian Cells

Summary

This study elucidates phagocytic processing of Godanti Bhasma (GB) particles in mammalian cells, characterizing their cellular uptake, vacuole dynamics, acidification, and degradation. These findings not only advance our understanding of fundamental phagocytosis mechanisms but also establish GB as a promising model system for developing novel therapeutic strategies.

Abstract

Phagocytosis is a vital cellular mechanism through which cells engulf and degrade foreign particles, pathogens, or debris, playing a key role in immune defense and the maintenance of tissue homeostasis. Disruptions in this process are associated with various diseases. To explore the complex events involved in the phagocytosis pathway, advanced smart particles and effective monitoring techniques are essential. Godanti Bhasma (GB), a traditional Indian medicine composed of bioactive calcium sulfate particles, is rapidly internalized by phagocytosis in mammalian cells, inducing significant cytoplasmic vacuolation. The key stages of GB-induced phagocytosis were evaluated here using flow cytometry (FC), live-cell imaging, and specific staining techniques. Flow cytometric analysis demonstrated the formation of phagocytic cup-like structures associated with particle internalization. Live-cell imaging enabled real-time observation of phagocytic processes, including particle uptake, vacuole formation, degradation of engulfed materials, and vacuolar turnover. Staining with neutral red and acridine orange was employed to assess vacuolar acidification. Interestingly, treatment with the lysosomal inhibitor BFA1 in GB-treated cells did not lead to vacuolation, as evidenced by the lack of neutral red uptake, emphasizing the requirement for an acidic environment for vacuolation to occur. These findings underscore the potential of GB-induced phagocytosis as a model to elucidate the sequential cellular events involved in this process, which is critical for understanding host-pathogen interactions, intracellular trafficking, and developing innovative therapeutic strategies for disorders related to phagocytosis.

Introduction

Phagocytosis is an essential cellular process carried out by tissue-resident professional phagocytes, such as macrophages and dendritic cells, as well as non-professional phagocytes, such as epithelial cells and fibroblasts, which engulf and degrade foreign particles. These cells internalize and eliminate foreign particles, pathogens, or cellular debris, contributing significantly to immune defense and tissue homeostasis¹. This process begins with the recognition of pathogens, debris, or particles through pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), which bind to pathogen-associated molecular patterns (PAMPs)

Protocol

1. Cell culture and seeding

1. Prepare complete DMEM medium supplemented with 10% heat-inactivated FBS and antibiotics (100 µg/mL penicillin, 100 µg/mL streptomycin). Sterilize all reagents and equipment before use.
2. Culture the 3T3-L1, HeLa, and RAW 264.7 cells in 25 cm² flasks and 35 mm culture dishes using DMEM. Maintain the cultures at 37 °C in a humidified incubator with 5% CO₂ and replace the medium every 48 h.
3. For subculturing, wash the cells 2x with PBS, then detach adherent cells using 500 µL of Trypsin-EDTA and incubate for 5 min. Add 1 mL of DMEM to neutralize the Trypsin....

Results

The study highlights key cellular responses to GB particles in mammalian cells. We used an imaging flow cytometer to investigate the internalization of GB particles in 3T3-L1 cells (Figure 1). The cells were exposed to particles for durations ranging from 30 min to 12 h. After 30 min, we observed the particles on the cell surface (Figure 1B), and the cell membrane began to fold inward at the attachment sites, indicating the initiation of internalization (

Discussion

Phagocytosis serves as a crucial immune defense mechanism, enabling the engulfment and subsequent elimination of particles, pathogens, and apoptotic cells. To understand the processes of phagosome formation and maturation, smart biocompatible particles combined with advanced imaging techniques are essential for studying dynamic events such as the vacuolar progression, acidification, and degradation of engulfed materials^18,19. We employed GB, a biocompatible model.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25 cm2 Culture Flasks	NEST, India	707001
96- and 6-well plates	Tarsons India Pvt. Ltd., Kolkata, India; Thermo Scientific, India	980040; 140675
Acridine orange	SRL, India	82407
Antibiotics, and Trypsin-EDTA solution	High Media, India	628242; 655111
Cell Lines- HeLa, 3T3-L1 and RAW-264.7	National Centre For Cell Science (NCCS), Pune, Maharastra
culture dishes (35mm)	SPL Lifesciences, India	101350
Dulbecco’s modified Eagle medium (DMEM)	MP Biomedicles, India	1033126
Fetal bovine serum (FBS)	High Media, India	651257
Flow cytometer	Millipore, USA	Amnis	Amnis Imaging Flow Cytometers,
Fluorscent Microscope	DiGi Lab, India	Model: DiGi 110 Infinity
Godanti Bhasma	Patanjali Ayurved	240002
Neutral Red dye	CDH, India	593630
Time lapse microscope	NanoEnTek Inc.  Seoul, Korea.	JuLi	JuLi smart fluorescent cell analyzer,
Time lapse microscope	LEEDZ MICRO IMA. UK	S.No. LIM225552358A58	EA PRIME Inverted microscope